Lated residueMembershipEnrichmentFIG. 3. Dynamics on the rapamycin-regulated phosphoproteome. A, identification of considerably
Lated residueMembershipEnrichmentFIG. 3. Dynamics in the rapamycin-regulated phosphoproteome. A, identification of drastically regulated phosphorylation web pages. The histogram shows the distribution of phosphorylation web site SILAC ratios for 1h rapamycincontrol (1hctrl) as well as the distribution of unmodified peptide SILAC ratios (red). The cutoff for regulated phosphorylation web-sites was determined according to two common deviations from the median for unmodified peptides. Unregulated web-sites are shown in black, and regulated sites are shown in blue. The numbers of down-regulated and up-regulated phosphorylation internet sites is indicated. B, the bar chart shows the distribution of phosphorylation sites into seven clusters, whereMolecular Cellular Proteomics 13.-7 -6 -5 -4 -3 -2 -1 0 1 2 3 four 5 6494Phosphorylation and Ubiquitylation Dynamics in TOR Signalingbehavior working with a fuzzy c-means algorithm (Figs. 3B and 3C) (40, 48). Regulated phosphorylation web sites had been clustered into six distinct profiles depending on the temporal behavior of those web pages. Distinct associations of GO terms within every cluster (Fig. 3D and supplemental Figs. S2H 2M) indicated that phosphorylation internet sites with distinct temporal profiles have been involved in the regulation of various biological processes. Cluster 1 incorporated web-sites that showed decreased phosphorylation over the time period of our experiment. This cluster integrated GO terms which include “signal transduction,” “ubiquitinprotein ligase activity,” and “positive regulation of gene expression” (supplemental Fig. S2H). Consistent with this, it encompassed identified regulated phosphorylation web sites including Thr142 in the transcriptional activator Msn4, which has been shown to reduce in response to osmotic anxiety (49), and Ser530 on the deubiquitylase Ubp1, a known Cdk1 substrate (50). This cluster also integrated various other fascinating proteins, like Gcd1, the subunit on the translation initiation aspect eIF2B; Pol1, the catalytic subunit in the DNA polymerase I -primase complex; Swi1, the transcription element that activates transcription of genes expressed at the MG1 phase of your cell cycle; and Atg13, the regulatory subunit of your Atg1p signaling complex that stimulates Atg1p SDF-1 alpha/CXCL12, Human kinase activity and is needed for vesicle formation for the duration of autophagy and cytoplasm-to-vacuole targeting. In contrast, cluster 6 contained websites at which phosphorylation improved more than the time period of our experiment. This cluster was PDGF-BB, Mouse (His) enriched in GO terms associated to nutrient deprivation, including “cellular response to amino acid starvation,” “amino acid transport,” “autophagy,” and “autophagic vacuole assembly” (supplemental Fig. S2M). It included phosphorylation websites on proteins for instance Rph1, Tod6, Dot6, Stb3, and Par32, which have previously been shown to become hyperphosphorylated after rapamycin therapy (51). Clusters 4 and five showed increases and decreases in phosphorylation, respectively, suggesting that these phosphorylation sites are possibly regulated as a consequence of modifications downstream of TOR inhibition, by way of example, by regulating the activity of downstream kinases and phosphatases upon rapamycin therapy. Clusters 2 and 3 contained internet sites at which the directionality of phosphorylation dynamics switched more than time, suggesting that these sites could be topic to a feedback regulation or controlled by a complex regulatory system. IceLogo (41) was utilized to analyze sequence motifs within the regulated phosphorylation internet site clusters (Fig. 3E). TOR kinase includes a.
